The development of recombinant DNA techniques has opened up the possibility of making specific changes in an organism's genetic makeup. Alteration of genetic endowment can lead to the development of useful strains of microorganisms, and more productive varieties of domesticated plants and animals. For example, insect resistant plants have recently been produced by the addition of a bacterial gene which instructs the recipient plants cells to produce a protein toxic to certain types of insects (see Vaeck, et al., Nature 328:33 (1987)). Manipulation of plant species through genetic engineering will become an important complement to classical breeding techniques in the development of plant varieties with new traits, such as improved nutritional quality, productivity, disease resistance, and drought and salinity tolerance.
Genetic engineering involves two basic processes: (1) isolation and propagation of new or altered genes (molecular cloning), and (2) the introduction of these genes into the recipient organism in a form that allows the introduced genetic information to be read (i.e., expressed) and transmitted to successive generations. The basic techniques of molecular cloning are well established, but the necessary tools for accomplishing the efficient introduction and expression of new genetic information in higher eukaryotic organisms are still limited.
Two general approaches are used to introduce new genetic information into cells, a procedure commonly referred to as "genetic transformation." One approach is to introduce the new genetic information as part of another DNA molecule, referred to as a "vector," which can be maintained as an independent unit (i.e., an episome) apart from the chromosomal DNA molecule(s). Episomal vectors contain all the necessary DNA sequence elements required for DNA replication and maintenance of the vector within the cell. Many episomal vectors are available for use in bacterial cells (for example, see Maniatis, T., et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, 1982)). However, only a few episomal vectors that function in higher eukaryotic cells have been developed. The available higher eukaryotic episomal vectors are based on naturally occurring viruses and most function only in mammalian cells. In higher plant systems there are no known double-stranded DNA viruses that replicate through a double-stranded intermediate upon which an episomal vector can be based. Although an episomal plant vector based on the Cauliflower Mosaic Virus has been developed, its capacity to carry new genetic information is limited (Brisson et al., Nature 310:511 (1984)).
The other general method of genetic transformation involves integration of the introduced DNA sequences into the recipient cell's chromosomes which permits the new information to be replicated and partitioned to the cell's progeny as a part of the natural chromosomes. The most common form of integrative transformation is called "transfection" and is frequently used in mammalian cell culture systems. Transfection involves introduction of relatively large quantities of deproteinized DNA into cells. The introduced DNA usually is broken and joined together in various combinations before it is integrated at random sites into the cell's chromosome (see, for example Wigler, et al., Cell 11:223 (1977)). A common problem with this procedure is the rearrangement of introduced DNA sequences (see Shingo, K., et al., Mol. Cell. Biol. 6:1787 (1986)). A more refined form of integrative transformation can be achieved by exploiting naturally occurring viruses that integrate into the host's chromosomes as part of their life cycle (e.g., retroviruses) (see Cepko, C., et al., Cell 37:1053 (1984)).
The most common genetic transformation method used in higher plants is based on the transfer of bacterial DNA into plant chromosomes that occurs during infection by the phytopathogenic soil bacterium Agrobacterium (see Nester et al., Ann. Rev. Plant Phys. 35:387-413 (1984)) . By substituting genes of interest for the naturally transferred bacterial sequences (called T-DNA), investigators have been able to introduce new DNA into plant cells. However, even this more "refined" integrative transformation system is limited in three major ways. First, DNA sequences introduced into plant cells using the Agrobacterium T-DNA system are frequently rearranged (see Jones, et al., Mol. Gen. Genet. 207:478 (1987)). Second, the expression of the introduced DNA sequences varies between individual transformants (see Jones et al., EMBO J. 4:2411-2418 (1985)). This variability is presumably caused by rearranged sequences and the influence of surrounding sequences in the plant chromosome (i.e., position effects). A third drawback of the Agrobacterium T-DNA system is the reliance on a "gene addition" mechanism: the new genetic information is added to the genome (i.e., all the genetic information a cell possesses) but does not replace information already present in the genome. While gene addition is suitable for many applications, the ability to actually replace a specific gene with an altered copy via homologous recombination (i.e., recombination between DNA of the same or a similar sequence) would be extremely useful. Gene replacement in mammalian cells using a transfection protocol has been attempted, but the procedure is inefficient (approximately 1 out of every 1000 transformed cells underwent a gene replacement event in Thomas et al., Cell 44:419 (1986); see also Smithies, et al., Nature 317:230 (1985)).
The present invention discloses linear episomal transformation vectors, based on natural chromosomes, that can replicate and be stably maintained in higher plant cells. These artificial plant chromosome vectors will provide a versatile tool for genetic transformation of plant species and solve many of the problems associated with present DNA transformation technology. In addition, development of artificial plant chromosome vectors will facilitate the construction of artificial chromosomes that can function in other higher eukaryotic cells.
Artificial chromosomes are man-made linear DNA molecules constructed from essential cis-acting DNA sequence elements that are responsible for the proper replication and partitioning of natural chromosomes (see Murray et al., Nature 301:189-193 (1983)). These essential elements are: (1) Autonomous Replication Sequences (ARS) (have properties of replication origins, which are the sites for initiation of DNA replication), (2) Centromeres (site of kinetochore assembly and responsible for proper distribution of replicated chromosomes at mitosis and meiosis), and (3) Telomeres (specialized structures at the ends of linear chromosomes that function to stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule).
At present, these essential chromosomal elements have been isolated only from lower eukaryotic species. ARSs have been isolated from the unicellular fungi Saccharomyces cerevisiae (brewer's yeast) and Schizosaccharomyces pombe (see Stinchcomb et al., Nature 282:39- 43 (1979) and Hsiao et al., J. Proc. Natl. Acad. Sci. USA 76:3829-3833 (1979)). ARSs behave like replication origins allowing DNA molecules that contain the ARS to be replicated as an episome after introduction into the cell nuclei of these fungi. Although plasmids containing these sequences replicate, they do not segregate properly.
Kinetochores are complex nucleo-protein structures, located at the centromeres, responsible for the proper partitioning of the chromosomes during mitosis and meiosis. The DNA component of the kinetochore, or centromeric DNA, provides the cis-acting signals specifying the location of kinetochore assembly, and controlling sister chromatic separation at mitotic and meiotic anaphase.
Functional centromeric (CEN) sequences have been purified from S. cerevisiae (see Clark et al., Nature 287:504-509 (1980) and Stinchcomb et al., J. Molec. Biol. 158:157-179 (1982)). Episomes carrying the yeast CEN sequences display proper segregation into daughter yeast cells during mitosis and meiosis, in contrast to ARS plasmids lacking a centromere.
The best characterized centromeric DNAs originate from the budding yeast Saccharomyces cerevisiae. Clarke and Carbon, Ann Rev Genet 19:29-56 (1985). The DNA region required for centromere function in S. cerevisiae is approximately 120 bp long and is composed of three conserved domains: CDEI, an 8 bp element [(A/G)TCAC(A/G)TG], CDEII, an extremely [.about.90%] AT-rich region of approximately 80 bp, and CDEIII, a 25 bp element [TGTTT(A/T)TGNTTTCCGAAANNNNAAA]. The molecular structure of centromeric DNAs from the fission yeast Schizosaccharomyces pombe have also been characterized. Several classes of S. pombe moderately repeated DNA elements have been identified which are found only in the centromere regions. These centromere-specific repetitive elements have been designated dg (3.8 kb), dh (4 kb), and yn by Yanagida and co-workers (Nakaseko et al., Embo. J. 5.:1011-1021 (1986); Nakaseko et al., Nuc. Acid Res. 15:4705-4715 (1987)), and K (6.4 kb), L (6 kb), and B (1 kb) by Carbon and his colleagues (Clarke et al., PNAS 83:8253-8257 (1986); Fishel et al., Mol. Cell Biol. 8:754-763 (1988)). The dg element has an AT-rich region and a 600 bp domain containing numerous small direct repeat motifs. Similarly, the dh element has an overall AT content approaching 70% and contains many short direct repeats. No nucleotide similarities to the S. cerevisiae CDEs have been found in the S. pombe elements.
Attempts to demonstrate that the S. pombe centromere-specific repetitive elements can function individually as centromeres have been unsuccessful. However, large restriction fragments (65 to 150 kb) carrying the entire fission yeast centromere regions of chromosome 1 or 3 function as centromeres when introduced into acentric episomes (Hahnenberger et al., PNAS USA 86:577-581 (1989)). These results indicate that either fission yeast centromeres are large composite structures that cannot be subdivided, or the functional fission yeast centromere element has not yet been identified.
In contrast to the detailed studies done in S. cerevisiae and S. pombe, essentially nothing is known about the molecular structure of functional centromeric DNA of higher eukaryotes. Ultrastructural studies indicate that higher eukaryotic kinetochores are large structures (e.g., mammalian kinetochore plates are approximately 0.3 .mu.m in diameter) which possess multiple microtubule attachment sites (reviewed in Rieder, Int. Rev. Cytol. 79:1-58 (1982)). It is therefore likely that the centromeric DNA regions of these organisms will be corresponding large.
Telomeres, the last chromosomal element in lower eukaryotes to be cloned, are thought to be involved in the priming of DNA replication at the chromosome end (see, for example, Blackburn et al., Ann. Rev. Biochem. 53:163-194 (1984)). This is because conventional DNA polymerases are template dependent, synthesize DNA in the 5' to 3' direction, and require an oligonucleotide primer to donate a 3' OH group. When this primer is removed, unreplicated single-stranded gaps arise; most of these gaps can be filled in by priming from 3' OH groups donated by newly replicated strands located at the 5' end of the gap. However, the unreplicated gaps which lie next to the extreme 5' end of the DNA duplex cannot be primed in this manner. Consequently, telomeres must provide an alternative priming mechanism.
Telomeres are also responsible for the stability of chromosomal termini. Telomeres act as "caps," suppressing the recombinogenic properties of free, unmodified DNA ends (see Blackburn, supra). This reduces the formation of damaged and rearranged chromosomes which arise as a consequence of recombination-mediated chromosome fusion events.
Telomeres may also contribute to the establishment or maintenance of intranuclear chromatin organization through their association with the nuclear envelope (see, for example, Fussell, C. P., Genetica 62:192-201 (1984)).
Telomeric or telomeric-like DNA sequences have been cloned from several lower eukaryotic organisms, principally protozoans and yeast. The ends of the Tetrahymena linear DNA plasmid have been shown to function like a telomere on linear plasmids in S. cerevisiae (see Szostak, J. W., Cold Spring Harbor Symp. Quant. Biol. 47:1187-1194 (1983)). A telomere from the flagellate Trypanosoma has been cloned (see, for example, Blackburn et al., Cell 36:447-457 (1984). A yeast telomeric sequence has been identified (see, for example, Shampay et al., Nature 310:154-157 (1984)).
None of the essential components, including the telomeres, however, function in higher eukaryotic systems. For example, there have been numerous attempts to isolate ARSs from other eukaryotes by selecting for pieces of DNA that will serve as an ARS in yeast. While such DNA fragments can be readily identified, they do not promote extrachromosomal replication in cells from the donor organism.
DNA molecules carrying ARSs that function in yeast cells do not promote extrachromosomal replication of these molecules in mouse cells (see Roth et al., Mol. Cell. Biol. 3:1898-1908 (1983)). An ARS sequenced from cultivated tomato, which operates in yeast, fails to function in tomato cells (see Zabel, P., "Toward the Construction of Artificial Chromosomes for Tomato," In: Molecular Form and Functions of the Plant Genome, (Plenum Press (1985) and Jongsma et al., Plant Molec. Biol. 8:383-394 (1987)). Similarly, yeast CEN sequences do not function when introduced into mouse or Aspergillus chromosomes (Boylan, et al., Mol. Cell. Biol. 6:3621 (1986)). In addition, telomeres from the protozoan Tetrahymena do not function in cells of the vertebrate Xenopus (Yu, et al., Gene 56:313 (1987)). Finally, although researchers were able to show that a S. pombe chromosome can replicate at a reduced efficiency in mouse cells, the centromeres of this lower eukaryote apparently do not function in the higher eukaryote nucleus (see Allshire et al., Cell 50:391-403 (1987)).
Artificial chromosomes have been constructed in yeast using the three cloned essential chromosomal elements. Murray et al., Nature 305:189-193 (1983), disclose a cloning system based on the in vitro construction of linear DNA molecules that can be transformed into yeast, where they are maintained as artificial chromosomes. These artificial yeast chromosomes contain cloned genes, replicators, centromeres and telomeres but have an impaired centromeric function in short (less than 20 kb) artificial chromosomes.
The ability to construct artificial chromosomes that function in yeast, however, does not teach one skilled in the art how to construct an artificial chromosome using the essential elements which will function in higher eukaryotes. The cells of higher eukaryotic organisms differ from yeast cells in ways that place many further demands on the functioning of their genes. The amounts of DNA in higher eukaryotes is large, varying between species in ways that are not yet completely understood. They have more DNA and their genomes are composed of different classes of sequences: true gene regions interrupted by numerous and lengthy introns, structural sequences necessary for chromosome sorting during cell division, and various kinds of repetitive sequences.
Lower eukaryotic systems were the only source of the artificial chromosome essential elements until the teaching of the present invention. It is clear that the construction of an efficient, functional artificial plant chromosome requires the isolation of the three essential elements from plant chromosomes, and this novel methodology and the elements derived therefrom are taught by the present invention.